Recombinant DNA technology has now introduced the possibility of producing, for example, pharmacologically-active proteins and peptides in microorganisms. Furthermore, it is possible to introduce changes by means of gene cloning such that the resulting polypeptides or proteins have improved or modified biological activity or stability as compared to the native gene product. However, prior to biological testing and clinical use, it is essential that the peptides or proteins should be purified to a very high degree in order to remove contaminating bacterial proteins, nucleic acids and endotoxins which may cause deleterious side effects. Therefore, there is a need for enhanced methods of purifying proteins produced using recombinant DNA techniques.
Currently, there are numerous methods available to purify peptides and proteins, e.g. affinity, ion-exchange, hydrophobic and molecular sieve chromatography, (see, for example, Williams, B. L., and Wilson, K., Principles and Techniques of Practical Biochemistry, (1975), Edward Arnold, London, 28-123). In order to achieve pure product in high yields and at reasonable cost, considerable development of such methods is necessary. Furthermore, at the moment, different methods must be developed and optimised for each new product. Even small changes in amino acid composition may alter the purification properties such that a modified purification procedure will need to be developed.
A further difficulty in the development of new products by recombinant DNA technology is the assay of the product. Many of the proteins and peptides have no enzymatic activity and may only be determined by either the in vitro or in vivo biological activity thereof. Such assays tend to be inaccurate and time consuming, while purification strategies require large numbers of highly accurate assay results. Immuno-assays based on the highly specific recognition of a protein by an antibody may provide such accurate and rapid assays, (see, for example, Eisen, H. N., Immunology, (1974), Harper and Row, U.S.A., 395-396). However, the raising of antisera to a protein is best achieved by inoculating animals with purified antigen and considerable expertise and time needs to be spent on this task. Also, not only would new antisera need to be raised for each new recombinant product, but because of the high specificity of these antibodies, even small modifications in the amino acid sequence may alter the binding of the product to the antibody and reduce the accuracy of the results.
One approach has been to fuse cloned peptides with a native bacterial protein, e.g. .beta.-galactosidase (.beta.-gal) and .beta.-lactamase (see, for example, Davis A. R., et al, Proc. Natl. Acad. Sci. U.S.A., (1981), 78, No. 9, 5376-5380; and published European Patent Application No. 35384). Hybrids may then acquire all of the properties of native protein, e.g. convenient assay, established purifications and, in the latter case, secretion from the host cell. However, in the case of .beta.-galactosidase, (.beta.-gal), which is a high molecular weight tetramer, the correct association of the .beta.-gal hybrid subunits may be altered or prevented by the tertiary structure of the hybrid. Although this does not occur when low molecular weight peptides are fused to .beta.-gal, there is no reason to assume that larger and structurally more complex hybrid proteins will still allow the correct association of subunits to form a fully active enzyme. Without subunit association, recombinants contained the fused polypeptide would not be identified by the .beta.-gal assay. In a similar manner, the alteration in secondary or tertiary structure of a .beta.-lactamase fused protein may prevent secretion thereof.
For clinical use, the cloned peptide or protein must be cleaved from the hybrid. Chemical cleavage at methionine residues has been described, but this is of limited use for most peptides and proteins, (see, for example, Goeddel, D. V. et al, Proc. Natl. Acad. Sci. U.S.A., (1979), 76, No. 1, 106-110). To this end, it has been suggested that, by introducing the correct peptide sequence, an endopeptidase might be used to specifically cleave the .beta.-gal protein from the desired peptide or protein, (see, for example, published European Patent Application No. 35384). For this approach to work, not only must this cleavage site be unique in the cloned protein or peptide, but also the folding of the entire fused protein must be such that the cleavage site is available to the endopeptidase. Such fused proteins would also share few similarities with the native endopeptidase substrate and the rate of cleavage may be considerably reduced. Furthermore, such endopeptidases could leave amino acids from the cleavage site on the protein of interest thereby making the protein unsuitable for many purposes.
Published European Patent Application No. 35384 related to DNA sequences coding for amino acid sequences which contain specific cleavage sites. These DNA sequences could be attached to a cloned DNA coding sequence. According to this reference, particularly the amino terminus of an expressed protein may be provided with a removable terminal sequence having distinctive physical properties which are useful for purification. Here it was important that the junction be provided with a cleavage site for an endopeptidase. In an attempt to approach the desired specificity, this prior art advocated the use of extended recognition sites for unusual enzymes. Of course, this procedure would have to be adapted to each protein and subject to the above limitations.
On the contrary, the present improved approach does not depend in the same way on the structure of the product. By virture of the use of an exopeptidase, the problem of simultaneous cleavage of the product is obviated without the need for complicated recognition sites for unusual enzymes. More importantly, unlike an endopeptidase, an exopeptidase will not hydrolyze the polypeptide product internally. The present system enjoys a further advantage in that the possibility of assay of the product is provided, which is not foreshadowed in the prior art. In the present case, attention is particularly directed to the carboxy terminus.
The present invention discloses a surprisingly useful process requiring a charged amino acid polymer and an exopeptidase that selectively removes the polymer and that does not harm the desired polypeptide product. The present invention includes any terminal amino acid polymer and the present invention also incudes the use of any exopeptidase, including both aminopeptidases and carboxypeptidases. The polymer may be at either (or both) the amino or carboxy terminals of the desired polypeptide product. Unlike the prior art the present invention allows via genetic engineering the attachment of an easily isolated polypeptide to a protein of interest followed by the selective removal of the attached polypeptide without harm to the protein of interest. In addition, the attached polypeptide serves as an easily quantitated tag reducing the requirement for expensive and difficult bioassays of the protein or its activity. A structural gene is defined as any gene coding for a polypeptide.